This invention relates to macrocyclic compounds and libraries of these compounds. The macrocyclic compounds of the invention have a plurality of nitrogenous sites that are derivatized by reaction with active forms of reactant compounds. The reactant compounds, upon covalently binding to the macrocyclic nitrogenous substrates, introduce diversity into the macrocyclic compounds. The reactant compounds are selected to be compounds that have, aside from a group capable of reacting with a nitrogenous species, a further functional group thereon that gives each individual compound at least one property that renders it diverse as compared to the other reactant compounds. Hence the incoming reactant compound bearing a chemical functional moiety imparts diversity to the macrocyclic compound and, upon bonding with the macrocycle, its residue can be referred to as a pendant chemical functional group.
The addition of the chemical functional groups at the several nitrogenous sites on the macrocyclic compound yields a macrocyclic compound having a unique set of properties. These properties include the overall global shape, the conformational space, electron density, dipole moment and ability of the compound to interact with enzyme pockets and other binding sites and other similar properties. Combinatorialized libraries of the macrocycles are synthesized having various permutations and combinations of the several chemical functional groups at the nitrogenous sites. Such synthesis is effected at each of the nitrogenous sites by presenting each nitrogenous site with several of the reactant compounds such that combinatorial mixtures are obtained. The libraries are deconvoluted to yield unique macrocyclic compounds. Preferred macrocycles of the invention have cyclophane-like structures.
The chemical functional groups on the macrocyclic compounds of the invention provide for binding of the compounds to proteins, including enzymes, nucleic acids, lipids and other biological targets. In preferred embodiments, the compounds of the invention act as inhibitors of pathogens such as virus, mycobacterium, bacteria (gram negative and gram positive), protozoa and parasites; as inhibitors of ligand-receptor interactions such as PDGF (platelet derived growth factor), LTB4 (leukotriene B4), IL-6 and complement C5A; as inhibitors of protein/protein interactions including transcription factors such as p50 (NFxcexaB protein) and fos/jun; as inhibitors of enzymes such as phospholipase A2; and for the inhibition of cell-based interactions including ICAM induction (using inducers such as IL1-xcex2, TNF and LPS). In other preferred embodiments, the compounds of the invention are used as diagnostic reagents, including diagnostic reagents in the tests for each of the above noted systems, and as reagents in assays and as probes. In even further preferred embodiments, the compounds of the invention are used as metal chelators and contrast agent carriers. In even further preferred embodiments, the compounds of the invention are used as herbicides and insecticides.
Traditional processes of drug discovery involve the screening of complex fermentation broths and plant extracts for a desired biological activity or the chemical synthesis of many new compounds for evaluation as potential drugs. The advantage of screening mixtures from biological sources is that a large number of compounds are screened simultaneously, in some cases leading to the discovery of novel and complex natural products with activity that could not have been predicted otherwise. The disadvantages are that many different samples must be screened and numerous purifications must be carried out to identify the active component, often present only in trace amounts. On the other hand, laboratory syntheses give unambiguous products, but the preparation of each new structure requires significant amounts of resources. Generally, the de novo design of active compounds based on the high resolution structures of enzymes has not been successful.
It is, thus, now widely appreciated that combinatorial libraries are useful per se and that such libraries and compounds comprising them have great commercial importance. Indeed, a branch of chemistry has developed to exploit the many commercial aspects of combinatorial libraries.
In order to maximize the advantages of each classical approach, new strategies for combinatorial deconvolution have-been developed independently by several groups. Selection techniques have been used with libraries of peptides (Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G. and Schoofs, P. G., J. Immun. Meth. 1987, 102, 259-274; Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. T. and Cuervo, J. H., Nature, 1991, 354, 84-86; Owens, R. A., Gesellchen, P. D., Houchins, B. J. and DiMarchi, R. D., Biochem. Biophys. Res. Commun., 1991, 181, 402-408; Doyle, M. V., PCT WO 94/28424; Brennan, T. M., PCT WO 94/27719); nucleic acids (Wyatt, J. R., et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 1356-1360; Ecker, D. J., Vickers, T. A., Hanecak, R., Driver, V. and Anderson, K., Nucleic Acids Res., 1993, 21, 1853-1856); nonpeptides and small molecules (Simon, R. J., et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 9367-9371; Zuckermann, R. N., et al., J. Amer. Chem. Soc., 1992, 114, 10646-10647; Bartlett, Santi, Simon, PCT WO91/19735; Ohlmeyer, M. H., et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 10922-10926; DeWitt, S. H., Kiely, J. S., Stankovic, C. J., Schroeder, M. C. Reynolds Cody, D. M. and Pavia, M. R., Proc. Natl. Acad. Sci. USA, 1993, 90, 6909-6913; Cody et al., U.S. Pat. No. 5,324,483; Houghten et al., PCT WO 94/26775; Ellman, U.S. Pat. No. 5,288,514; Still et al., PCT WO 94/08051; Kauffman et al., PCT WO 94/24314; Carell, T., Wintner, D. A., Bashir-Hashemi, A. and Rebek, J., Angew. Chem. Int. Ed. Engel., 1994, 33, 2059-2061; Carell, T., Wintner, D. A. and Rebek, J., Angew. Chem. Int. Ed. Engel., 1994, 33, 2061-2064; Lebl, et al., PCT WO 94/28028). We have developed certain nitrogen coupled chemistries that we utilized to prepare a class of compounds we refer to as xe2x80x9coligonucleosides.xe2x80x9d We have described these compounds in previous patent applications including published PCT applications WO 92/20822 (PCT US92/04294) and WO 94/22454 (PCT US94/03313). These chemistries included amine linkages, hydroxylamine linkages, hydrazino linkages and other nitrogen based linkages.
A review of the above references reveals that the most advanced of these techniques are those for selection of peptides and nucleic acids. Several groups are working on selection of heterocycles such as benzodiazepines. With the exception of Rebek et al., scant attention has been given to combinatorial discovery of other types of molecules. No combinatorial discovery approaches have been reported for non-linear (non-peptide, non-nucleic acid) macromolecules.
The majority of the techniques reported to date involve iterative synthesis and screening of increasingly simplified subsets of oligomers. Monomers or sub-monomers that have been utilized include amino acids, amino acid-like molecules, i.e. carbamate precursors, and nucleotides, both of which are bifunctional. Utilizing these techniques, libraries have been assayed for activity in either cell-based assays, or for binding and/or inhibition of purified protein targets.
A technique, called SURF(trademark) (Synthetic Unrandomization of Randomized Fragments), involves the synthesis of subsets of oligomers containing a known residue at one fixed position and equimolar mixtures of residues at all other positions. For a library of oligomers four residues long containing three monomers (A, B, C), three subsets would be synthesized (NNAN, NNBN, NNCN, where N represents equal incorporation of each of the three monomers). Each subset is then screened in a functional assay and the best subset is identified (e.g. NNAN). A second set of libraries is synthesized and screened, each containing the fixed residue from the previous round, and a second fixed residue (e.g. ANAN, BNAN, CNAN). Through successive rounds of screening and synthesis, a unique sequence with activity in the functional assay can be identified. The SURF(trademark) technique is described in Ecker, D. J., Vickers, T. A., Hanecak, R., Driver, V. and Anderson, K., Nucleic Acids Res., 1993, 21, 1853-1856. The SURF(trademark) method is further described in PCT patent application WO 93/04204, the entire disclosure of which is herein incorporated by reference.
The combinatorial chemical approach that has been most utilized to date, utilizes an oligomerization from a solid support using monomeric units and a defined connecting chemistry, i.e. a solid support monomer approach. This approach has been utilized in the synthesis of libraries of peptides, peptoids, carbamates and vinylogous peptides connected by amide or carbamate linkages or nucleic acids connected by phosphate linkages as exemplified by the citations in previous paragraphs above. A mixture of oligomers (pool or library) is obtained from the addition of a mixture of activated monomers during the coupling step or from the coupling of individual monomers with a portion of the support (bead splitting) followed by remixing of the support and subsequent splitting for the next coupling. In this monomeric approach, each monomeric unit would carry a tethered letter, i.e., a functional group for interaction with the target. Further coupling chemistry that allows for the insertion of a tethered letter at a chemically activated intermediate stage is referred to as the sub-monomer approach.
The diversity of the oligomeric pool is represented by the inherent physical properties of each monomer, the number of different monomers mixed at each coupling, the physical properties of the chemical bonds arising from the coupling chemistry (the backbone), the number of couplings (length of oligomer), and the interactions of the backbone and monomer chemistries. Taken together, these interactions provide a unique conformation for each individual molecule.
There remains a need in the art for molecules which have fixed preorganized geometry that matches that of targets such as proteins and enzymes, nucleic acids, lipids and other targets. The backbone of such molecules should be rigid with some flexibility, and such molecules should be easy to construct in solution or via automated synthesis on solid support.
Unsymmetrical N-substituted polyazamacrocycles are disclosed in Gu, K., et al., Tetrahedron Lett., 1989, 30, 1323-1326.
Synthesis of diaza[7]-,[8]-,[9]-,[10]-(2,6)pyridinophanes are disclosed in Krakowiak, K., Polish Journal of Chemistry, 1986, 60, 277-281.
It is an object of the invention to provide macrocyclic compounds for diagnostic, research, and therapeutic use.
It is a further object of the invention to provide macrocyclic compounds that have a plurality of nitrogenous sites for introducing chemical functional groups into the macrocycle to provide xe2x80x9cdiversityxe2x80x9d to the basic macrocycle.
It is yet another object of the invention to provide methods for preparing libraries of diversified macrocyclic compounds.
It is a still further object of the invention to provide libraries of combinatorialized macrocyclic compounds.
These and other objects will become apparent to persons of ordinary skill in the art from a review of the present specification and the appended claims.
The present invention provides novel cyclophane-like compounds and macrocyclic compounds comprising cyclic structures that are interrupted with at least one aromatic, alicyclic or heterocyclic ring system having two bridgehead atoms, each bridgehead atom being connected to at least one bridge containing at least two nitrogenous moieties bearing chemical functional groups.
In certain embodiments, each chemical functional group is, independently, of the structure:
xe2x80x83xe2x80x94Txe2x80x94L;
where T is a single bond, a methylene group or a group having the structure
xe2x80x94{[CR1R2]mxe2x80x94(R5)xe2x80x94[CR1R2]nxe2x80x94[C(R6)]pxe2x80x94(E)xe2x80x94}qxe2x80x94
wherein:
R6 is xe2x95x90O, xe2x95x90S, xe2x95x90NR3;
R5 and E, independently, are a single bond, CHxe2x95x90CH, Cxe2x89xa1C, O, S, NR3, SO2, or C6-C14 aryl;
each R1, R2 and R3 is, independently, H, alkyl or haloalkyl having 1 to about 10 carbon atoms; alkenyl having 2 to about 10 carbon atoms; alkynyl having 2 to about 10 carbon atoms; or aryl having 6 to about 14 carbon atoms;
m and n, independently, are zero to 5;
p is zero or 1;
q is 1 to about 10; and
L is, H, C2-C10 alkyl or substituted alkyl, C2-C10 alkenyl or substituted alkenyl, C2-C10 alkynyl or substituted alkynyl, C4-C7 carbocyclic alkyl, or substituted C4-C7 carbocyclic alkyl, alkenyl or alkynyl carbocyclic, or substituted alkenyl or alkynyl carbocyclic, or C6-C14 aryl or substituted aryl where the substituent groups are selected from hydroxyl, amino, alkoxy, alcohol, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, or alkynyl groups; an ether having 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms; a nitrogen, sulfur or oxygen containing heterocycle; a metal coordination group; a conjugate group; halogen; hydroxyl (OH); thiol (SH); keto (Cxe2x95x90O); carboxyl (COOH); amide (CONR1); amidine (C(xe2x95x90NH) NR1R2); guanidine (NHC(xe2x95x90NH) NR1R2); glutamyl R3OOCCH (NR1R2)(CH2) 2C (xe2x95x90O)xe2x80x94); nitrate (ONO2); nitro (NO2); nitrile (CN); trifluoromethyl (CF3); trifluoromethoxy (OCF3); O-alkyl; S-alkyl; NH-alkyl; N-dialkyl; O-aralkyl; S-aralkyl; NH-aralkyl; amino (NH2); azido (N3); hydrazino (NHNH2); hydroxylamino (ONH2); sulfoxide (SO); sulfone (SO2); sulfide (Sxe2x80x94); disulfide (Sxe2x80x94S); silyl; a nucleosidic base; an amino acid side chain; a carbohydrate; a drug; or a group capable of hydrogen bonding.
In certain preferred embodiments of the present invention, compounds are provided of the structure: 
wherein:
xx is 0-4;
R4 is a single bond connecting Q and A; or a group having the formula: 
xe2x80x83wherein
each Z is, independently, H, hydroxyl, amino, thiol, acyl, protected hydroxyl, protected amino, protected thiol, protected acyl, or Nxe2x80x94(Txe2x80x94L)2;
each r is, independently, from 0 to about 8;
each J is, independently, N, O, S, or a heterocyclic ring system;
each t is, independently, 0 or 1;
L is H, C1-C10 alkyl or substituted alkyl, C2-C10 alkenyl or substituted alkenyl, C2-C10 alkynyl or substituted alkynyl, C4-C7 carbocyclic alkyl, alkenyl or alkynyl or substituted carbocyclic, or C6-C14 aryl or substituted aryl where the substituent groups are selected from hydroxyl, amino, alkoxy, alcohol, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, or alkynyl groups; an ether having 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms; a nitrogen, sulfur or oxygen containing heterocycle; a metal coordination group; a conjugate group; halogen; hydroxyl (OH); thiol (SH); keto (Cxe2x95x90O); carboxyl (COOH); amide (CONR1); amidine (C(xe2x95x90NH)NR1R2); guanidine (NHC(xe2x95x90NH)NR1R2); glutamyl R3OOCCH(NR1R2)(CH2)2C(xe2x95x90O)xe2x80x94); nitrate (ONO2); nitro (NO2); nitrile (CN); trifluoromethyl (CF3); trifluoromethoxy (OCF3); O-alkyl; S-alkyl; NH-alkyl; N-dialkyl; O-aralkyl; S-aralkyl; NH-aralkyl; amino (NH2); azido (N3); hydrazino (NHNH2); hydroxylamino (ONH2); sulfoxide (SO); sulfone (SO2); sulfide (Sxe2x80x94); disulfide (Sxe2x80x94S); silyl; a nucleosidic base; an amino acid side chain; a carbohydrate; a drug; or a group capable of hydrogen bonding;
T is a single bond, a methylene group or a group having the structure:
xe2x80x94{[CR1R2]mxe2x80x94(R5)xe2x80x94[CR1R2]nxe2x80x94[C(R6)]pxe2x80x94(E)xe2x80x94}qxe2x80x94
each R6 is, independently, xe2x95x90O, xe2x95x90S, xe2x95x90NR3;
each R5 and E is, independently, a single
bond, CHxe2x95x90CH, Cxe2x89xa1C, O, S, NR3, or C6-C14 aryl;
each R1, R2 and R3 is, independently, H, alkyl or haloalkyl having 1 to about 10 carbon atoms; alkenyl having 2 to about 10 carbon atoms; alkynyl having 2 to about 10 carbon atoms; or aryl having 6 to about 14 carbon atoms;
each m and n is, independently, zero to 5;
each p is, independently, zero or 1;
q is 1 to about 10;
each Q is an aromatic, alicyclic, or heterocyclic ring system;
each A is a group having the structure:
xe2x80x94Mxe2x80x94(X)dxe2x80x94(CH2)exe2x80x94"Brketopenst"xe2x80x94(X)dxe2x80x94(CH2)ee"Brketclosest"zxe2x80x94(X)axe2x80x94Gxe2x80x94
xe2x80x83wherein
each X is, independently, xe2x80x94N(TL)xe2x80x94, xe2x80x94N(TL)xe2x80x94Oxe2x80x94,xe2x80x94N(TL)xe2x80x94SO2xe2x80x94, xe2x80x94N(TL)xe2x80x94SOxe2x80x94, xe2x80x94N(TL)Sxe2x80x94, xe2x80x94N(TL)xe2x80x94C(O)xe2x80x94, xe2x80x94(TL)NHxe2x80x94C(O)xe2x80x94, xe2x80x94N(TL)xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94N(TL)xe2x80x94(CO)xe2x80x94NRxe2x80x94, or xe2x80x94N (TL)xe2x80x94(CH2)xxe2x80x94Oxe2x80x94;
M and G, independently, are xe2x80x94(CH2)exe2x80x94, xe2x80x94N(TL)xe2x80x94(CH2)exe2x80x94, xe2x80x94Oxe2x80x94(CH2)e, xe2x80x94Sxe2x80x94(CH2)exe2x80x94, xe2x95x90Nxe2x80x94, xe2x80x94N(R)xe2x80x94, O, S, xe2x80x94C(O)xe2x80x94;
each e is, independently, from 1 to about 5;
each ee is, independently, from 1 to about 5;
each d is, independently, from 0 to about 2;
z is from 1 to about 9;
a is from 0 to about 2; and
with the proviso that not more than three of the chemical functional groups, represented by xe2x80x94Txe2x80x94L, of each of the groups Q, are p-toluenesulfonyl or H and that at least two of the chemical functional groups are different.
Combinatorial synthetic strategies offer the potential to generate and screen libraries comprising extremely large numbers of compounds and identify individual molecules with desired properties. In certain embodiments of the invention, cyclophane-like compounds are provided that comprise part of a library of such compounds bearing varied chemical functional groups. Further, cyclophane-like compounds are provided bearing at least three different chemical functional groups thereon.
In accordance with some embodiments of the present invention, a library of compounds is provided comprising a plurality of macrocyclic molecules having an identical macrocyclic structure, a plurality of at least two different types of nitrogenous moieties and a plurality of chemical functional groups covalently bonded to the nitrogenous moieties.
In accordance with one embodiment of the present invention, a library of compounds is provided comprising a plurality of macrocyclic molecules having an identical macrocyclic structure, a plurality of nitrogenous moieties and a plurality of chemical functional groups covalently bonded to the nitrogenous moieties.
Methods of the present invention are useful for generating a library of macromolecules. These methods comprise selecting a macromolecule having at least one ring system, one bridge for each ring system connected to at least one bridgehead atom thereof, and having a plurality of nitrogenous moieties located within the macromolecule. These methods further comprise selection and reaction of a plurality of chemical functional group reactants for introduction of chemical functional groups on the nitrogenous moieties.